Genes encoding plant transcription factors

ABSTRACT

Identification of a gene from monocotyledonous plants such as rice, which codes for a transcription factor specific to a stress tolerant gene and provision of a novel environmental stress tolerant plant using the gene. From the rice genome, a gene, which binds to a cis element existing upstream of the gene encoding a stress responsive protein and for a transcription factor to activate the transcription of the gene, is identified. Further, the gene of the transcription factor is used to transform a plant, thereby improving tolerance against environmental stresses such as low temperature, dehydration, and salt stresses.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.10/302,382, filed Nov. 22, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a protein, which regulates rice-derivedenvironmental stress tolerance, a gene encoding the same, and a methodfor utilizing the same.

2. Prior Art

Plants possess tolerance mechanisms to cope with various types ofenvironmental stresses in nature such as dehydration, high temperature,freezing, or salt stress. In the production of plants having suchenvironmental stress tolerance, techniques have been heretofore used forgenetically selecting and mating strains which are dehydration, salt, orlow temperature tolerant. However, these techniques require long periodsof time to select, and also have low success rates.

On the other hand, as the stress tolerance mechanism is elucidated at amolecular level, stress tolerant plants have been produced usingbiotechnological techniques. For example, it has been shown that stressproteins such as LEA proteins, water channel proteins, or synthetasesfor compatible solutes are induced in cells when they are exposed tostress, thereby protecting the cells from such stress. Thus, researchhas been attempted in which genes such as LEA proteins of barley ordetoxification enzymes of tobacco, or genes of synthetases forosmoregulatory substances (e.g., sugar, proline, or glycinebetaine) areintroduced into host plants. Research using genes encoding w-3 fattyacid desaturase of Arabidopsis thaliana, the D9-desaturase of blue-greenalgae, or the like, which are modification enzymes of the cellularmembrane lipid, has also been attempted. In the above researches, a genewas bound to the 35S promoter of cauliflower mosaic virus and introducedinto a plant. The level of stress tolerance of the recombinant plantwas, however, low and unstable. Thus, none of these was put to practicaluse.

On the other hand, stress tolerance mechanism is found to be intricatelyassociated with several genes (Plant Physiol., 115: 327-334 (1997)).Accordingly, research in which a gene encoding a transcription factorwhich simultaneously activates the expression of the genes is introducedinto a plant, thereby enhancing the plant's stress tolerance, has beenattempted (The Plant Cell, 10: 1-17 (1998)). However, when several genesare simultaneously activated, the energy of the host plant becomesdirected towards the generation of the gene product or intracellularmetabolism resulting from the gene product. Accordingly, the growth ofthe plant itself deteriorates or becomes retarded.

In contrast, the present inventors had isolated the genes DREB1A,DREB1B, DREB1C, DREB2A, and DREB2B encoding the transcription factorswhich bind to a stress responsive element and specifically activate thetranscription of genes located downstream of the element fromArabidopsis thaliana (Japanese Patent Application No. 10-22847,Laying-Open (kokai) No. 2000-60558). They reported that introduction andoverexpression of the genes in a plant enabled impartment of stresstolerance without causing retardation of a plant (Japanese PatentApplication No. 10-292348, Laying-Open (kokai) No. 2000-116260).

Arabidopsis thaliana is classified as a dicotyledonous plant while majorcrops such as rice, maize, and wheat are classified as monocotyledonousplants. Dicotyledonous plants are relatively different frommonocotyledonous plants from the viewpoint of plants evolution. It hasbeen shown that the DREB1A gene of Arabidopsis thaliana functions wellin monocotyledonous plants, but not as well in dicotyledonous plants.Thus, if a DREB-homologous gene derived from the monocotyledonous plantcan be isolated, environmental stress tolerance can be more efficientlytransmitted to monocotyledonous plants thereby.

SUMMARY OF THE INVENTION

An object of the present invention is to identify from amonocotyledonous plant such as rice, a gene which codes for atranscription factor specific to a stress tolerant gene, and to providea novel environmental stress tolerant plant using the same gene.

The present inventors have conducted concentrated studies in order toattain the above object. As a result, they had succeeded in identifyingall the DREB-homologous genes from the rice genome. They also found thatintroduction of the genes into other plants significantly enhanced theirenvironmental stress tolerance. This has led to the completion of thepresent invention.

More specifically, the present invention provides the following (1) to(12).

(1) An isolated gene comprising the following DNA (a) or (b):

-   -   (a) DNA which comprises the nucleotide sequence as shown in SEQ        ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID        NO: 9; or    -   (b) DNA which hybridizes with the DNA comprising a nucleotide        sequence, which is complementary to the DNA comprising the        nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ        ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 under stringent        conditions and which codes for a protein that regulates the        transcription of genes located downstream of a stress responsive        element.

(2) An isolated gene encoding the following protein (c) or (d):

-   -   (c) a protein which comprises the amino acid sequence as shown        in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or        SEQ ID NO: 10; or    -   (d) a protein which comprises the amino acid sequence having        deletion, substitution, or addition of one or several amino        acids in the amino acid sequence as shown in SEQ ID NO: 2, SEQ        ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 and which        regulates the transcription of genes located downstream of a        stress responsive element.

(3) The gene according to (1) or (2) above, wherein the stress isdehydration stress, low temperature stress, or salt stress.

(4) The following recombinant protein (c) or (d):

-   -   (c) a protein which comprises the amino acid sequence as shown        in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or        SEQ ID NO: 10; or    -   (d) a protein which comprises the amino acid sequence having        deletion, substitution, or addition of one or several amino        acids in the amino acid sequence as shown in SEQ ID NO: 2, SEQ        ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 and which        regulates the transcription of genes located downstream of a        stress responsive element.

(5) The protein according to (4) above, wherein the stress isdehydration stress, low temperature stress, or salt stress.

(6) A recombinant vector comprising the gene according to any one of (1)to (3) above.

(7) A transformant transformed with the recombinant vector according to(6) above.

(8) The transformant according to (7) above, wherein the host is aplant.

(9) The transformant according to (7) above, wherein the host is amonocotyledonous plant.

(10) A method for producing a protein which regulates the transcriptionof genes located downstream of a stress responsive element, wherein thetransformant according to (8) or (9) above is cultured in a medium andthe protein is recovered from the resultant culture product.

(11) A method for determining stress levels in plants, wherein thetranscription levels of the gene according to any one of (1) to (3)above in plant bodies are determined.

(12) A method for improving the stress tolerance of plants byintroducing the gene according to any one of (1) to (3) above into theplants.

The present invention provides a stress tolerant transcription factorderived from monocotyledonous plants and a gene encoding thistranscription factor. Use of the gene according to the present inventionenables the more efficient transmission of stress tolerance to crops,i.e., monocotyledonous plants.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawings executedin color. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a structure of the OsDREB protein (A: OsDREB1, B: OsDREB2).

FIG. 2 shows an amino acid sequence of a DREB1-homologous protein(OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D; rice, BCBF3; barley, DREB1A;Arabidopsis thaliana, ACRE111B; tobacco).

FIG. 3 shows an amino acid sequence of a DREB2-homologous protein(OsDREB2A; rice, DREB2A: Arabidopsis thaliana, ORCA1; Catharanthusroseus).

FIG. 4 shows the result of a gel shift assay.

FIG. 5(A) shows the structure of a plasmid used in transactivation, andFIG. 5(B) shows a ratio of introduction efficiency by transactivation(GUS/LUC).

FIG. 6 shows the result of Northern blotting analysis of expression ofOsDREB genes.

FIG. 7 shows the result of analysis of stress tolerant gene expressionin a recombinant Arabidopsis thaliana.

FIG. 8 shows the result of analysis of stress tolerant gene expressionin a recombinant rice.

FIG. 9 shows salt stress tolerance of a recombinant plant (Arabidopsisthaliana) into which OsDREB1A and DREB1A have been introduced.

DETAILED DESCRIPTION OF THE INVENTION

This specification includes part or all of the contents as disclosed inthe specification of Japanese Patent Application No. 2001-358268, whichis a priority document of the present application.

The gene according to the present invention is a gene derived from ricegenome having tolerance improving mechanisms against environmentalstresses such as low temperature, dehydration, or salt stress.

The gene of the present invention is “an isolated gene encoding atranscription factor which binds to a cis element located upstream ofgenes encoding stress responsive proteins expressed in response toenvironmental stresses such as low temperature, dehydration, or saltstress, thereby activating the transcription of the genes”. Specificexamples of the above cis element include dehydration-responsive element(DRE), abscisic acid-responsive element (ABRE), and lowtemperature-responsive element. The protein encoded by the gene of thepresent invention functions to activate the transcription of geneslocated downstream of the above-mentioned stress responsive elements(DRE or the like).

The gene according to the present invention can be identified as, forexample, described below.

1. Identification of the Gene of the Present Invention

The gene according to the present invention can be screened based onhomology with a known gene having homologous functions, that is, a geneencoding a transcription factor specific to a stress tolerant gene of aplant. mRNA and cDNA libraries of rice or a rice genomic library may beprepared and may be subjected to screening. Alternatively, an existingdatabase of rice DNA may be subjected to screening.

A. Screening of Gene Library

(1) Preparation of mRNA and cDNA Libraries

At the outset, mRNA and cDNA libraries are prepared as follows.

As a source of mRNA, parts of the plant body of rice such as leaves,stems, roots, or flowers, or the plant body as a whole may be used.Alternatively, a plant body obtained by sowing rice seeds on a solidmedium such as GM medium, MS medium, or #3 medium and growing themaseptically may be used. The source may be a callus or a cultured ricecell which was aseptically grown, and the variety thereof is notparticularly limited as long as the cell contains mRNA of the gene ofinterest. Further, since the gene to be screened is expressed inresponse to environmental stress, plants that are exposed to lowtemperature stress (e.g. 10 to −4° C.), salt stress (e.g. 150 to 250 mMNaCl), or dehydration stress (e.g. dehydrated state) can also bepreferably used.

For example, mRNA is prepared as follows. Rice plant bodies, which hadbeen grown hydroponically to low temperature stress, dehydration stress,or salt stress are exposed and then freezed with liquid nitrogen. Thefrozen plant bodies are ground in a mortar. From the resultant groundmaterial, crude RNA fraction is extracted and prepared by the glyoxalmethod, the guanidine thiocyanate-cesium chloride method, the lithiumchloride-urea method, the proteinase K-deoxyribonuclease method, or thelike. From this crude RNA fraction, poly(A)+RNA (mRNA) can be thenobtained by the affinity column method using oligo dT-cellulose or polyU-Sepharose carried on Sepharose 2B or by the batch method. Theresultant mRNA may further be fractionated by sucrose density gradientcentrifugation or the like.

Further, a cDNA library can be produced using the thus obtained mRNA asa template. For example, single-stranded cDNA is synthesized using anoligo(dT) primer or random primer, and a reverse transcriptase in acommercially available kit (e.g. ZAP-cDNA Synthesis Kit: Stratagene).Then, double-stranded cDNA is synthesized from the resultantsingle-stranded cDNA. Subsequently, an adaptor containing a suitablerestriction site is added to the resultant double-stranded cDNA, whichis then inserted into a cloning site of a lambda phage vector. Theresultant DNA is packaged using Gigapack III Gold packaging extract(Stratagene) or the like and infected into an E. coli host, and thenamplified. Thereafter, phage particles are recovered and stored.

(2) Preparation of Genomic Library

For example, the preparation of a genomic library using a lambda phagevector is carried out in the following manner. As a source of DNA, partsof the rice plant body such as leaves, stems, roots, or flowers, or theplant body as a whole may be used as long as the tissue contains DNA.The plant body is pulverized in the presence of liquid nitrogen, and DNAis extracted by the CTAB method, the benzyl chloride method, or thelike. The resultant DNA is partially decomposed with the restrictionenzyme Sau3AI and then fractionated by NaCl density gradientultracentrifugation or the like to recover 10 to 20 kb fragments. Thesefragments are inserted into the BamHI cleavage site of lambda phagevectors such as λEMBL3 and λFIX II. Thereafter, packaging is carried outusing Gigapack III Gold packaging extract (Stratagene) or the like,followed by infection into an E. coli host. The amplified phageparticles are then recovered and stored.

(3) Screening of Library

A library can be screened in the following manner.

A DNA fragment as a probe is prepared based on a sequence in a highlyconserved region of, for example, a known gene encoding a transcriptionfactor specific to a stress tolerant gene of a plant, such as the DREBgene derived from Arabidopsis thaliana (DREB1A gene: SEQ ID NO: 11,DREB2A gene: SEQ ID NO: 12, DREB1B gene: SEQ ID NO: 13, DREB1C gene: SEQID NO: 14, DREB2B gene: SEQ ID NO: 15). The probe DNA may be amplifiedby PCR using two primers with approximately 15 bp to 25 bp which aredesigned based on the sequence of each side of the highly conservedregion so as to amplify said region. When the highly conserved region isshort and insufficient as a probe, a primer may be designed to amplifyseveral highly conserved regions together with the regions adjacentthereto.

Using the above probe, a cDNA library or genomic library is screened byplaque hybridization or colony hybridization.

B. Screening Using Gene Database

Important sequences (highly conserved regions or regions deduced to havedesired function) of, for example, a known gene that encodes atranscription factor specific to a stress tolerant gene of a plant suchas DREB gene derived from Arabidopsis thaliana (DREB1A gene, DREB1Bgene, DREB1C gene, DREB2A genes, DREB2B gene) are specified.Subsequently, homology search on an existing gene database is conductedbased on the specified sequence. The genetic data to be searched may beEST or a full-length gene. Homology search can be carried out using ananalytical software such as BLAST or FASTA on databases of GenBank orDDBJ. Preferably, the object of detection is a gene encoding an aminoacid sequence which has an especially high homology with an amino acidsequence in the highly conserved region or a region deduced to havedesired function, consequently a sequence conserving an amino acidsequence that is essential for the function of a protein. Based on theresultant sequence, a primer is designed, and PCR is carried out usinguncloned cDNA (RT-PCR), a cDNA library, genomic DNA, or a genomiclibrary as a template, thereby obtaining the gene of interest.Alternatively, a DNA fragment amplified by PCR is used as a probe and acDNA library or genomic library is screened to obtain the gene ofinterest.

C. Determination of Nucleotide Sequences

The entire nucleotide sequence of the cloned cDNA can be determined inaccordance with conventional methods. Nucleotide sequencing includes thechemical modification method of Maxam-Gilbert or the dideoxynucleotidechain termination method using M13 phage. Usually, sequencing is carriedout using an automated nucleotide sequencer (e.g., 377 DNA Sequencer,Perkin-Elmer).

Thus, OsDREB1A (SEQ ID NO: 1), OsDREB1B (SEQ ID NO: 3), OsDREB1C (SEQ IDNO: 5), OsDREB1D (SEQ ID NO: 7), and OsDREB2A (SEQ ID NO: 9) wereidentified as DREB-homologous genes derived from rice.

Also, OsDREB1A protein (SEQ ID NO: 2), OsDREB1B protein (SEQ ID NO: 4),OsDREB1C protein (SEQ ID NO: 6), OsDREB1D protein (SEQ ID NO: 8), andOsDREB2A protein (SEQ ID NO: 10), which were coded by the genes throughanalysis of ORFs of the genes, were identified.

The genes according to the present invention, however, are not limitedto genes comprising DNA as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Genes comprising DNA, which arehybridizable under stringent conditions with DNA comprising a nucleotidesequence that is complementary to the DNA comprising a nucleotidesequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, or SEQ ID NO: 9, are also genes of the present invention as longas they code for proteins that regulate the transcription of geneslocated downstream of a stress responsive element.

The term “stringent conditions” as used herein refers to parameters withwhich the art is familiar. Stringent conditions are sequence-dependentand will be different in different circumstances. Longer sequenceshybridize specifically at higher temperatures. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength andpH. The T_(m) is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target sequence hybridize to the target sequence at equilibrium.Nucleic acid hybridization parameters may be found in references whichcompile such methods, e.g., Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Second Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989. More specifically, stringentconditions, as used herein, refers, for example, to those conditions inwhich formamide concentration is 30-50%, temperature is 37 to 50° C.,and 6×SSC. Preferably, formamide concentration is 50%, temperature is42° C., and 6×SSC.

The genes of the present invention are genes encoding proteinscomprising amino acid sequences as shown in SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. Even though a proteincomprises an amino acid sequence having deletion, substitution, oraddition of one or several amino acids in the amino acid sequence asshown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQID NO: 10, genes encoding this protein is included as genes according tothe present invention as long as this protein can regulate thetranscription of genes located downstream of a stress responsiveelement. The term “several amino acids” preferably refers to 20 or fewerand more preferably 5 or fewer amino acids.

The protein according to the present invention is not limited to aprotein comprising an amino acid sequence as shown in SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. A protein whichcomprises an amino acid sequence having one or several amino acidsdeleted, substituted, or added in the amino acid sequence as shown inSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10is included as a protein according to the present invention as long asit can regulate the transcription of genes located downstream of astress responsive element. The term “several amino acids” preferablyrefers to 20 or fewer and more preferably 5 or fewer amino acids.

The introduction of mutation into the gene of the present invention maybe performed by conventional techniques such as the Kunkel method, theGapped duplex method or variations thereof using a mutation introducingkit [e.g. Mutant-K (Takara) or Mutant-G (Takara)] utilizingsite-directed mutagenesis or using an LA PCR in vitro Mutagenesis SeriesKit (Takara).

Once the nucleotide sequence for the gene of the present invention hasbeen determined, the gene of the present invention can be obtainedeither by chemical synthesis, by PCR using the cDNA or genomic DNA ofthe gene as a template, or by the hybridization of a DNA fragment havingthe above nucleotide sequence as a probe.

2. Analysis of the DRE Binding Ability and Transcription ActivatingAbility of the Proteins of the Present Invention

A. Analysis of the DRE Binding Ability

The ability of the protein according to the present invention to bind toDRE can be confirmed by gel shift assay [Urao, T. et al., Plant Cell5:1529-1539 (1993)] using a fusion protein composed of the protein, GST,and the like. The protein according to the present invention can beprepared by ligating the gene according to the present inventiondownstream of the glutathione-S-transferase (GST) coding region of aplasmid coding for GST gene (e.g. pGEX-4T-1 vector: Pharmacia) in amanner that the reading frames of the two genes coincide with eachother, culturing E. coli that has been transformed with the plasmidunder conditions which induce synthesis of the fusion protein, andpurifying the protein from the transformed E. coli.

Gel shift assay is a method for examining the interaction between DNAand a protein. A DRE-containing DNA fragment labeled with ³²P or thelike is mixed with the fusion protein described above and incubated, andthe resultant mixture is subjected to electrophoresis. After drying, thegel is autoradiographed to detect those bands which have migrated to theback as a result of the binding of the DNA fragment and the protein. Thespecific binding of the protein according to the present invention tothe DRE sequence can be confirmed by showing that the above-mentionedband is not detected when a DNA fragment containing a mutated DREsequence is used.

B. Analysis of Transcription Activating Ability

The transcription activating ability of the proteins of the presentinvention can be analyzed by a transactivation experiment using riceprotoplast system. For example, OsDREB1A cDNA is ligated to pBI221plasmid (Clontech) containing CaMV35 S promoter to construct an effectorplasmid. On the other hand, the DRE-containing DNA fragment is ligatedupstream of TATA promoter located upstream of a β-glucuronidase (GUS)gene to construct a reporter plasmid. Subsequently, these two plasmidsare introduced into rice protoplasts and then GUS activity is measured.If GUS activity is increased by the simultaneous expression of OsDREB1Aprotein, it is understood that OsDREB1A protein expressed in theprotoplasts is activating the transcription through the DRE sequence.

In the present invention, preparation of protoplasts and introduction ofplasmid DNA into the protoplasts may be performed by the method of Abelet al. [Abel, S. et al., Plant J. 5:421-427 (1994)]. In order tominimize experimental errors resulting from differences in plasmid DNAintroduction efficiencies, a plasmid in which luciferase gene is ligateddownstream of CaMV35S promoter may be introduced to protoplasts togetherwith the two plasmids described above, thus, β-glucuronidase activityagainst luciferase activity may be determined. Then, the determinedvalue may be taken to indicate transcription activating ability.β-glucuronidase activity can be determined by the method of Jefferson etal. [Jefferson, R. A. et al., EMBO J. 83:8447-8451 (1986)]; andluciferase activity can be determined using PicaGene Luciferase AssayKit (Toyo Ink).

3. Preparation of Recombinant Vectors and Transformants

A. Preparation of Recombinant Vectors

The recombinant vector of the present invention can be obtained byligating (inserting) the gene of the present invention to (into) anappropriate vector. The vector into which the gene of the presentinvention is to be inserted is not particularly limited as long as it isreplicable in a host. For example, plasmid DNA, phage DNA or the likemay be used. Plasmid DNA includes plasmids for E. coli hosts such aspBR322, pBR325, pUC118, and pUC119; plasmids for Bacillus subtilis hostssuch as pUB110 and pTP5; plasmids for yeast host such as YEp13, YEp24,and YCp50; and plasmids for plant cell host such as pBI221 and pBI121.Phage DNA includes λ phage and the like. Further, animal virus vectorsuch as retrovirus or vaccinia virus; or insect virus vector such asbaculovirus may also be used.

In order to insert the gene of the present invention into a vector, forexample, a method may be employed in which the purified DNA is cleavedwith an appropriate restriction enzyme and then inserted into therestriction site or the multi-cloning site of an appropriate vector DNAfor ligation to the vector. The gene of the present invention should beincorporated into the vector in such a manner that the function of thegene is expressed. For this purpose, in addition to a promoter and thegene of the present invention, those containing cis elements such asenhancer, a splicing signal, poly(A) addition signal, selection marker,ribosome binding sequence (SD sequence) or the like can be ligated tothe vector of the present invention, if so desired. Examples ofselection marker are dihydrofolate reductase gene, ampicillin tolerancegene, neomycin tolerance gene, or the like.

B. Preparation of Transformants

The transformant of the present invention can be obtained by introducingthe recombinant vector of the present invention into a host so that thegene of interest can be expressed. The host is not particularly limitedas long as the gene of the present invention can be expressed therein.Specific examples of the host include Escherichia bacteria such as E.coli; Bacillus bacteria such as Bacillus subtilis; Pseudomonas bacteriasuch as Pseudomonas putida; Rhizobium bacteria such as Rhizobiummeliloti; yeasts such as Saccharomyces cerevisiae, Schizosaccharomycespombe; plant cell strains established from Arabidopsis thaliana,tobacco, maize, rice, carrot, etc. or protoplasts prepared from suchplants; animal cells such as COS cells, CHO cells; or insect cells suchas Sf9 cells and Sf21 cells.

When a bacterium such as E. coli is used as the host, the recombinantvector of the present invention is capable of autonomous replicationinside the host and, at the same time, it is preferably composed of apromoter, a ribosome binding sequence, the gene of the presentinvention, and a transcription termination sequence. The vector may alsocontain a gene to regulate the promoter. Escherichia coli strains suchas HMS174 (DE3), K12, or DH1 may be used. Bacillus subtilis strains suchas MI 114 or 207-21 may be used.

Any promoter may be used as long as it is able to direct the expressionof the gene of interest in a host such as E. coli. For example, an E.coli- or phage-derived promoter such as trp promoter, lac promoter,P_(L) promoter, or P_(R) promoter may be used. An artificially designedand altered promoter such as tac promoter may also be used. Methods forintroducing the recombinant vector into a bacterium are not particularlylimited, and examples thereof include a method using calcium ions[Cohen, S. N. et al., Proc. Natl. Acad. Sci., USA, 69:2110-2114 (1972)]and electroporation.

When yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,or Pichia pastoris is used as the host, the promoter is not particularlylimited, and any promoter may be used as long as it is able to directthe expression of the gene of interest in yeast. For example, gal1promoter, gal 10 promoter, heat shock protein promoter, MFα1 promoter,PH05 promoter, PGK promoter, GAP promoter, ADH promoter, or AOX1promoter can be used.

A method for introducing the recombinant vector into yeast is notparticularly limited, and examples thereof include electroporation[Becker, D. M. et al., Methods Enzymol., 194:182-187 (1990)], thespheroplast method [Hinnen, A. et al., Proc. Natl. Acad. Sci., USA,75:1929-1933 (1978)], and the lithium acetate method [Itoh, H., J.Bacteriol., 153:163-168 (1983)].

When a plant cell is used as the host, for example, cell strainsestablished from rice, maize, wheat, Arabidopsis thaliana, tobacco,carrot, etc. or protoplasts prepared from such plants, the promoter tobe used is not particularly limited as long as it is able to direct theexpression of the gene of interest in plants. Examples thereof include35S RNA promoter of cauliflower mosaic virus, rd29A gene promoter, andrbcS promoter.

A method for introducing the recombinant vector into a plant includesthe method of Abel et al. using polyethylene glycol [Abel, H. et al.,Plant J. 5:421-427 (1994)] and electroporation. When an animal cell isused as the host, for example, simian COS-7 or Vero cells, Chinesehamster ovary cells (CHO cells), mouse L cells, rat GH3 cells, human FLcells, or the like, SRα promoter, SV40 promoter, LTR promoter, CMVpromoter or the like may be used. The early gene promoter of humancytomegalovirus or the like may also be used.

To introduce the recombinant vector into an animal cell, for example,electroporation, the calcium phosphate method, lipofection, or the likemay be used. When an insect cell is used as the host, for example, Sf9cells, Sf21 cells, or the like, the calcium phosphate method,lipofection, electroporation, or the like may be used.

4. Production of the Proteins According to the Present Invention

The protein of the present invention is a protein having an amino acidsequence encoded by the gene of the present invention; or a proteinwhich has an amino acid sequence having at least one amino acid mutationin the above-described amino acid sequence and is able to regulate thetranscription of genes located downstream of a stress responsiveelement.

The protein of the present invention can be obtained by culturing thetransformant in a medium and recovering the protein from the resultantculture product. The term “culture product” means any of the followingmaterials: culture supernatant, cultured cells, cultured microorganisms,or disrupted cells or microorganisms. The transformant of the presentinvention in a medium is cultured by conventional methods for culturinga host.

As a medium for culturing the transformant obtained from a microorganismhost such as E. coli or yeast, either a natural or synthetic medium maybe used as long as it contains carbon sources, nitrogen sources, andinorganic salts assimilable by the microorganism and is capable ofefficient culture of the transformant. When a plant cell is used as thehost, vitamins such as thiamine and pyridoxine can be added to themedium, if necessary. When an animal cell is used as the host, serumsuch as RPMI1640 can be added to the medium, if necessary.

Examples of carbon sources include: carbohydrates such as glucose,fructose, sucrose, and starch; organic acids such as acetic acid andpropionic acid; and alcohols such as ethanol and propanol. Examples ofnitrogen sources include: ammonia; ammonium salts of inorganic ororganic acids such as ammonium chloride, ammonium sulfate, ammoniumacetate, and ammonium phosphate; other nitrogen-containing compounds;peptone; meat extract; and corn steep liquor.

Examples of inorganic substances include: monopotassium phosphate,dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, iron(I) sulfate, manganese sulfate, copper sulfate, andcalcium carbonate. Usually, the culture is carried out under aerobicconditions (such as shaking culture or aeration agitation culture) atapproximately 30 to 37° C. for approximately 6 hours to 3 days. Duringthe culture, the pH is maintained at approximately 7.0 to 7.5. The pH isadjusted with an inorganic or organic acid, an alkali solution, or thelike.

During the culture, an antibiotic such as ampicillin or tetracycline maybe added to the medium, if necessary. When a microorganism transformedwith an expression vector containing an inducible promoter is cultured,an inducer may be added to the medium, if necessary. For example, when amicroorganism transformed with an expression vector containing Lacpromoter is cultured, isopropyl-β-D-thiogalactopyranoside (IPTG) or thelike may be added to the medium. When a microorganism transformed withan expression vector containing trp promoter is cultured, indoleacrylicacid (IAA) or the like may be added to the medium.

Usually, the culture is carried out in the presence of 5% CO₂ atapproximately 30 to 37° C. for approximately 6 hours to 3 days. Duringthe culture, an antibiotic such as kanamycin or penicillin may be addedto the medium if necessary. After the culture, the protein of thepresent invention is extracted by disrupting the cultured microorganismor cell if the protein is produced in the microorganism or cell. If theprotein of the present invention is secreted outside of themicroorganism or cell, the culture fluid may be used for the followingsteps as it is or subjected to centrifugation to remove themicroorganism or cells. Thereafter, conventional biochemical techniquesused for isolating/purifying a protein, for example, ammonium sulfateprecipitation, gel chromatography, ion exchange chromatography, andaffinity chromatography, are employed independently or in an appropriatecombination to isolate and purify the protein of the present inventionfrom the above culture product.

5. Preparation of Transgenic Plants into which the Gene of the PresentInvention has been Introduced

A transgenic plant tolerant to environmental stresses, in particular,low temperature, freezing, and dehydration stresses, can be produced byintroducing DNA encoding the protein of the present invention into ahost plant using genetic engineering techniques. A method forintroducing the gene of the present invention into a host plant includesindirect introduction such as the Agrobacterium infection method anddirect introduction such as the particle gun method, polyethylene glycolmethod, liposome method, and microinjection method. When theAgrobacterium infection method is used, the transgenic plant of thepresent invention can be produced by the following procedure.

A. Preparation of a Recombinant Vector to be Introduced into a Plant andTransformation of Agrobacterium

A recombinant vector to be introduced into a plant can be prepared bycleaving with an appropriate restriction enzyme DNA comprising the genesof the present invention, ligating an appropriate linker to theresultant DNA if necessary, and inserting the DNA into a cloning vectorfor the plant cell host. A binary vector type plasmid such aspBI2113Not, pBI2113, pBI101, pBI121, pGA482, pGAH, and pBIG, or anintermediate vector type plasmid such as pLGV23Neo, pNCAT, and pMON200may be used as cloning vectors.

When a binary vector type plasmid is used, the gene of interest isinserted between the border sequences (LB, RB) of the binary vector. Theresultant recombinant vector is amplified in E. coli. The amplifiedrecombinant vector is then introduced into Agrobacterium tumefaciensC58, LBA4404, EHA101, C58C1Rif^(R), EHA105, etc. by freeze-thawing,electroporation, or the like. The resultant Agrobacterium is used forthe transformation of the plant of interest.

In the present invention, the three-member conjugation method [NucleicAcids Research, 12:8711 (1984)] may also be used in addition to themethod described above to prepare an Agrobacterium containing the geneof the present invention for plant infection. Specifically,plasmid-containing E. coli comprising the gene of interest, helperplasmid-containing E. coli (e.g. pRK2013), and an Agrobacterium aremixed and cultured on a medium containing rifampicin and kanamycin.Thus, a zygote Agrobacterium for infecting plants can be obtained.

For the expression of a foreign gene and the like in a plant body, apromoter and a terminator for plants should be located upstream anddownstream of the structural gene, respectively. Specific examples ofpromoters which may be utilized in the present invention includecauliflower mosaic virus (CaMV)-derived 35S transcript [Jefferson, R. A.et al., The EMBO J. 6:3901-3907 (1987)]; the promoter for maizeubiquitin gene [Christensen, A. H. et al., Plant Mol. Biol. 18:675-689(1992)]; the promoter for nopaline synthase (NOS) gene; and the promoterfor octopin (OCT) synthase gene. Specific examples of useful terminatorinclude CaMV-derived terminators and NOS-derived terminators. Promotersand terminators are not limited to the above-mentioned as long as theyare known to function in plant bodies.

If the promoter used in a transgenic plant is a promoter responsible forthe constitutive expression of the gene of interest (e.g., CaMV 35Spromoter) and the use thereof has brought about delay in the growth orretardation of the transgenic plant, a promoter which directs transientexpression of the gene of interest (e.g., rd29A gene promoter) may beused. If necessary, an intron sequence, which enhances the expression ofthe gene of the present invention, may be located between the promotersequence and the gene. For example, the intron from maize alcoholdehydrogenase (Adh1) [Genes & Development 1:1183-1200 (1987)] may beintroduced.

In order to efficiently select transformed cells of interest, it ispreferable to use an effective selection marker gene in combination withthe gene of the present invention. As the selection marker, one or moregenes, which are selected from kanamycin tolerance (NPTII) gene,hygromycin phosphotransferase (htp) gene which confers tolerance to theantibiotic hygromycin on plants, phosphinothricin acetyl transferase(bar) gene which confers tolerance to bialaphos, and the like, can beused. The gene of the present invention and the selection marker genemay be incorporated together into a single vector. Alternatively, twotypes of recombinant DNAs may be used which are incorporated intoseparate vectors.

B. Introduction of the Gene of the Present Invention into a Host

In the present invention, while the host for the transformant is notparticularly limited, it is preferably a plant. The plant may be anycultured plant cells, the entire plant body of a cultured plant, plantorgans (such as leaves, petals, stems, roots, rhizomes, or seeds), orplant tissues (such as epidermis, phloem, parenchyma, xylem, or vascularbundle). Plants are preferably monocotyledonous plants such as rice,maize, and wheat. When a cultured plant cell, plant body, plant organ orplant tissue is used as the host, the Agrobacterium infection method,particle gun method, or polyethylene glycol method can be employed tointroduce the DNA encoding the protein of the present invention totransform this host plant by introducing a vector into plant sections.Alternatively, a vector can be introduced into a protoplast byelectroporation to produce a transformed plant.

For example, when a gene is introduced into Arabidopsis thaliana by theAgrobacterium infection method, the step of infecting the plant with anAgrobacterium containing a plasmid comprising the gene of interest isessential. This step can be performed by the vacuum infiltration method[CR Acad. Sci. Paris, Life Science, 316:1194 (1993)]. Specifically,Arabidopsis thaliana is grown in a soil composed of equivalent portionsof vermiculite and perlite. The Arabidopsis thaliana is immerseddirectly in a culture fluid of an Agrobacterium containing a plasmidcomprising the gene of the present invention, placed in a desiccator,and then sucked with a vacuum pump to 65-70 mmHg. Then, the plant isallowed to stand at room temperature for 5-10 min. The plant pot istransferred to a tray, which is covered with a wrap to maintainhumidity. On the next day, the wrap is removed. The plant is grown inthat state to harvest seeds.

Subsequently, the seeds are sown on MS agar medium supplemented withappropriate antibiotics to select those individuals which have the geneof interest. Arabidopsis thaliana grown on this medium are transferredto pots and grown there. As a result, seeds of a transgenic plant intowhich the gene of the present invention has been introduced can beobtained. Generally, the genes are introduced into the genome of thehost plant in a similar manner. However, due to differences in thespecific locations on the genome into which the genes have beenintroduced, the expression of the introduced genes varies. Thisphenomenon is called “position effect.” By assaying transformants withDNA fragments from the introduced gene as a probe by Northern blotting,it is possible to select those transformants in which the introducedgene is expressed more highly.

The confirmation that the gene of interest is integrated in thetransgenic plant into which the gene of the present invention has beenintroduced and in the subsequent generation thereof can be made byextracting DNA from cells and tissues of those plants and detecting theintroduced gene by PCR or Southern analysis, which are conventionalmethods in the art.

C. Analysis of the Expression Level and Expression Site of the Gene ofthe Present Invention in Plant Issues

The expression level and expression site of a gene in a transgenic plantinto which the gene of the present invention has been introduced can beanalyzed by extracting RNA from cells and tissues of the plant anddetecting the mRNA of the introduced gene by RT-PCR or Northernanalysis, which are conventional methods in the art. Alternatively, theexpression level and expression site can be analyzed directly by Westernblotting or the like of the gene product of the present invention usingan antibody against the above product.

D. Changes in the mRNA Levels of Various Genes in a Transgenic Plantinto which the Gene of the Present Invention has been Introduced

It is possible to identify by Northern hybridization those genes whoseexpression levels are believed to have been changed as a result of theaction of the transcription factor of the present invention in atransgenic plant into which the gene of the present invention has beenintroduced.

For example, plants grown hydroponically or the like are givenenvironmental stress for a specific period of time (e.g. 1 to 2 weeks).Examples of environmental stresses include low temperature, dehydration,and salt stresses. For example, dehydration stress may be given byuprooting the plant from the hydroponic medium and drying it on a filterpaper for 10 minutes to 24 hours. Low temperature stress may be given byretaining the plant at 15 to −4° C. for 10 minutes to 24 hours. Saltstress can be given by, for example, replacing the hydroponic solutionwith a 50 to 500 mM NaCl solution and retaining the plant for 10 minutesto 24 hours.

Total RNAs are respectively prepared from a control plant, which wasgiven no stress, and from the plant, which was given environmentalstress, and the resultant total RNAs are subjected to electrophoresis.The expression patterns can be analyzed by Northern hybridization usingthe probe of the gene to be observed.

E. Evaluation of the Tolerance of the Transgenic Plant to EnvironmentalStresses

The tolerance to environmental stresses of the transgenic plant intowhich the gene of the present invention has been introduced can beevaluated by setting the transgenic plant in a pot containing a soilcomprising vermiculite, perlite and the like, exposing the plant tovarious environmental stresses, and examining the survival of the plant.Environmental stresses include low temperature, dehydration, and saltstresses. For example, tolerance to dehydration stress can be evaluatedby leaving the plant without watering for 2 to 4 weeks and thenexamining the survival. Tolerance to low temperature and freezingstresses can be evaluated by leaving the plant at 15 to −10° C. for 1 to10 days, growing it at 20 to 35° C. for 2 days to 3 weeks, and thenexamining its survival ratio. Tolerance to salt stress can be evaluatedby, for example, leaving the plant in 100 to 600 mM NaCl for 1 hour to 7days, growing it at 20 to 35° C. for 1 to 3 weeks, and then examiningits survival rate.

F. Determination of Stress Levels in Plants

The transcription of the gene according to the present invention isactivated by low temperature stress, dehydration stress, or salt stress.Therefore, determination of the transcription level of the gene of thepresent invention enables the assessment of the stress level such as lowtemperature, dehydration, or salt stress which the plant is subjectedto.

The transcription level of the gene according to the present inventioncan be determined by, for example, RNA gel blot analysis or quantitativePCR. A probe to be used in RNA gel blot analysis can be produced inaccordance with any conventional method based on the gene according tothe present invention and/or a 100-1000 bp region comprising specificsequence adjacent to the gene. A primer to be used in quantitative PCRcan be prepared by any conventional method based on the sequence in theregion encoding the gene of the present invention or the region adjacentthereto.

The above-described probe or primer may be used in a kit for determiningthe transcription level of the gene according to the present invention.

G. Others

In addition, the protein according to the present invention can beutilized by producing an antibody against the protein. The antibody maybe a polyclonal or monoclonal antibody. The method for producing anantibody is not particularly limited, and it can be carried out inaccordance with any conventional method [see, for example, Sambrook, Jet al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)].The antibody can be utilized in, for example, the detection of theprotein of interest by Western blotting or immunoprecipitation.

EXAMPLES

The present invention is described in more detail with reference to thefollowing examples, however, the scope of the present invention is notlimited to these.

Example 1 Screening of Rice OsDREB Gene

1. Homology Search Against Database

On the basis of the full-length amino acid sequences of DREB1A, DREB1B,DREB1C, DREB2A, and DREB2B genes as shown below, homology search wascarried out by BLAST against the database of rice DNA in GenBank.

As a result, four types of genes were discovered: 1 type (OsDREB1B) fromEST data, 2 types (OsDREB1C and OsDREB1D) from genome sequence data interms of the DREB1-homologous gene, and 1 type (OsDREB2A) from EST datain terms of the DREB2-homologous gene.

2. Search of cDNA Library

A. Preparation of cDNA Library

Rice seeds (Nipponbare) were grown hydroponically using distilled waterunder dark conditions at 25° C. for 15 days. The resulting plant bodieswere treated at 4° C. for 2 hours or 24 hours, uprooted from theincubator and dried on a filter paper for 10 hours, or treated with 250mM NaCl for 10 hours, followed by freezing with liquid nitrogen. TotalRNA was extracted from the frozen sample using the guanidinethiocyanate-cesium chloride method, and mRNA was prepared using theOligo(dt)-cellulose column. cDNA was synthesized using the resultantmRNA as a template and using HybriZAP-2.1 two-hybrid cDNA Gigapackcloning kit (Stratagene) and the cDNA was inserted and cloned in theEcoRI-XhoI cleavage site of HybriZAP-2.1 phagemid vector. This phagemidDNA was packaged using Gigapack III Gold packaging extract (Stratagene).The obtained lambda phage particles containing cDNA were used to infecthost E. coli, which were then amplified, and these were subsequentlyrecovered. The resulting phage suspension was then stored.

B. Search Using Probe

The sequence containing the ERF/AP2 domain and a conserved region on theN-terminal side of the OsDREB1D genome sequence obtained in (1) wasamplified by PCR to produce a probe. The cDNA library, which wasprepared in (1), was searched using this probe. As a result, a newDREB1-homologous cDNA (OsDREB1A) was obtained.

The EST clone corresponding to OsDREB1B was provided by the Rice GenomeResearch Projects. To amplify the full-length of the protein-codingregion, OsDREB1C and OsDREB1D were subjected to PCR using a primer thatwas designed on the basis of predictions from a genome sequence of atranscription initiation site and a termination codon.

The probe for searching the full-length cDNA of OsDREB2A was producedbased on the sequence of EST, thereby searching the cDNA library. Sincethe resultant cDNA clone was predicted to be of an incomplete length,5′RACE was carried out using DNA prepared from the cDNA library as atemplate to determine the full-length sequence. Based on this sequence,a primer for amplifying a full-length gene was designed and thefull-length gene was obtained by RT-PCR.

C. Nucleotide Sequencing

The nucleotide sequence of the cDNA of the resultant DREB-homologousgene was determined using 377 DNA sequencer (Perkin-Elmer). Further, theORF was analyzed to determine all the amino acid sequences.

3. Results:

As a result, nucleotide sequences for 5 types of OsDREB genes andcorresponding amino acid sequences of OsDREB proteins were identified.As the DREB protein derived from Arabidopsis thaliana, all the OsDREBproteins comprised regions which were deduced to be: the ERF/AP2 DNAbinding domain at the center, a nuclear localization signal at theN-terminus, and an acidic activation domain at the C-terminus (FIG. 1).

In FIG. 2 and FIG. 3, amino acid sequences of the DREB-homologousproteins from various plants were compared to one another to find highlyconserved sequences. Outline letters on colored backgrounds representhighly conserved regions.

Sequence numbers of nucleotide sequences and amino acid sequences ofeach OsDREB are as follows:

-   -   OsDREB1A: nucleotide sequence (SEQ ID NO: 1), amino acid        sequence (SEQ ID NO: 2);    -   OsDREB1B: nucleotide sequence (SEQ ID NO: 3), amino acid        sequence (SEQ ID NO: 4);    -   OsDREB1C: nucleotide sequence (SEQ ID NO: 5), amino acid        sequence (SEQ ID NO: 6);

OsDREB1D: nucleotide sequence (SEQ ID NO: 7), amino acid sequence (SEQID NO: 8);

-   -   OsDREB2A: nucleotide sequence (SEQ ID NO: 9), amino acid        sequence (SEQ ID NO: 10).

Example 2 Analysis of Ability of OsDREB Proteins to Bind to DRE

A fusion protein between glutathione-S-transferase (GST) and proteins ofOsDREB1A and OsDREB2A was prepared using E. coli. The resulting proteinwas then assessed by gel shift assay to inspect the proteins' bindingabilities to DRE.

The 477 bp DNA fragment located from position 69 to position 545 of thenucleotide sequence of OsDREB1A cDNA or the 489 bp DNA fragment locatedfrom position 334 to position 822 of the nucleotide sequence of OsDREB2AcDNA was amplified by PCR. Then, the amplified fragment was ligated tothe EcoRI-XhoI site of plasmid pGEX-4T-1 (Pharmacia). After theintroduction of this plasmid into E. Coli XL1-Blue MRF′, the E. coli wascultured in 500 ml of 2×YT medium (Molecular Cloning (1982), Cold SpringHarbor Laboratory Press). To this culture, 0.1 mM isopropylβ-D-thiogalactoside, which activates the promoter of plasmid pGEX-4T-1,was added to induce the synthesis of a fusion protein of OsDREB1A andGST.

The E. coli into which the protein had been induced was suspended in 18ml of buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 5 mM MgCl₂, 400 mMNaCl, 5% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mMdithiothreitol). Then, 1% Triton X-100 and 1 mM EDTA were added thereto.After the cells were disrupted by sonication, the disrupted material wascentrifuged at 20,000 g for 1 hour. Then, the protein was purified fromthe supernatant using glutathione-Sepharose (Pharmacia). The resultantfusion protein was incubated at room temperature for 20 minutes usingthe DRE sequence-containing 75 bp DNA fragment (SEQ ID NO: 16) labeledwith ³²P as a probe. This mixture was electrophoresed using 5%polyacrylamide containing 0.25×Tris-borate-EDTA at 120 V for 90 minutes.As a result of this gel shift assay, those bands which migrated to theback were detected. When the DNA fragment containing the mutated DREsequence was used as a probe, such bands were not detected. Thus, itbecame evident that OsDREB1A and OsDREB2A proteins specifically bind tothe DRE sequence (FIG. 4).

Example 3 Preparation of Transformant (Transgenic Plant)

1. Construction of Plant Plasmid

A. Preparation of OsDREB1A Gene Fragment

The 717 bp DNA fragment located from position 69 to position 785 of thenucleotide sequence of cDNA of the OsDREB1A gene was amplified by PCRusing the following primers. Thereafter, the amplified fragment wasligated to the BamHI cleavage site of the vector pBluescript SK(−)(Stratagene) to obtain the recombinant plasmid pSKOsDREB1A. ThispSKOsDREB1A was cleaved with BamHI to obtain approximately 700 bp DNAfragment containing OsDREB1A gene.

Forward: (SEQ ID NO: 17) 5′-GGGGATCCATGTGCGGGATCAAGCAGGAGATG-3′ Reverse:(SEQ ID NO: 18) 5′-GGGGATCCCTAGTAGCTCCAGAGTGGGAC-3′

B. Preparation of pBE2113Not, G-ubi, G35S-ShΔ

pBE2113Not (Plant Cell 10: 1391-1406(1998)), G-ubi, and G35S-ShΔ wereused as plasmids having promoter DNA. G-ubi and G35S-ShΔ were preparedas follows. At the outset, pBIG plasmid (Nucleic Acids Research 18: 203(1990)) was cleaved with BamHI, blunt-ended and ligated to delete theBamHI cleavage site. Thereafter the plasmid was cleaved with HindIII andEcoRI. The resultant fragment and an approximately 1.2 kb fragment,which was obtained by cleavage of pBE2113Not plasmid in the same manner,were ligated to each other, thereby preparing pBIG2113Not plasmid.

Subsequently, pBIG2113Not was cleaved with HindIII and BamHI and ligatedto a fragment of rd29A promoter (approximately 0.9 kb, NatureBiotechnology 17: 287-291 (1999)), which was cleaved in the same manner,thereby preparing pBIG29APHSNot plasmid. Further, this pBIG29APHSNotplasmid was cleaved with HindIII and SalI and then ligated to a fragmentof the ubiquitin gene (Ubi-1) promoter (approximately 2.0 kb, PlantMolecular Biology 18: 675-689 (1992)) of maize, which was cleaved in thesame manner, or to a fragment (approximately 1.6 kb, Proceeding NationalAcademy of Science USA 96: 15348-15353 (1999)) containing CaMV 35Spromoter of p35S-shΔ-stop and a part of the intron of a sucrose synthasegene (Sh1) of maize. Thus, G-ubi plasmid or G35S-shΔ plasmid wasprepared. pBE2113Not, G-ubi, and G35S-shΔ described above wererespectively cleaved with BamHI and ligated to the OsDREB1A genefragment using Ligation High (Toyobo Co., Ltd.). E. coli DH5α wastransformed using the thus obtained ligation product. After thetransformant was cultured, plasmid pBE35S:OsDREB1A, G-ubi: OsDREB1A, andG35S-ShΔ: OsDREB1A were respectively purified therefrom. Subsequently,the nucleotide sequences thereof were determined, and those havingOsDREB1A gene bound in the sense direction were selected.

C. Introduction into Agrobacterium

The plasmid pBE35S: OsDREB1A-containing E. coli DH5α, helper plasmidpRK2013-containing E. coli HB101, and Agrobacterium C58 were mixed andcultured on LB agar medium at 28° C. for 24 hours. Generated colonieswere scraped off and suspended in 1 ml of LB medium. This suspension (10μl) was coated on LB agar medium containing 100 mg/l rifampicilin and 20mg/l kanamycin and cultured at 28° C. for 2 days, thereby obtainingzygote Agrobacterium C58 (pBE35S: OsDREB1A). By electroporation, theplasmid G-ubi: OsDREB1A and plasmid G35S-ShΔ: OsDREB1A were separatelyintroduced into Agrobacterium EHA105, which were then washed with 10%glycerol after culturing. Thus, Agrobacterium EHA105 (G-ubi:OsDREB1A)and Agrobacterium EHA105 (G35S-shΔ: OsDREB1A) were prepared.

2. Gene Introduction into Arabidopsis thaliana by AgrobacteriumInfection

The zygote was cultured in 10 ml of LB medium containing 100 mg/lrifampicilin and 20 mg/l kanamycin at 28° C. for 24 hours. Subsequently,this culture fluid was added to 500 ml of LB medium and cultured for 24hours. The resultant culture fluid was centrifuged to remove the mediumand suspended in 500 ml of buffer for infection (2.3 g of Murashige andSkoog Plant Salt Mixture (Nihon Pharmaceutical Co., Ltd), 1 ml ofGamborg's vitamin solution, 50 g of sucrose, 200 μl of L-77 (NipponUnicar Co., Ltd.), and 10 μg of 6-benzylaminopurine, per liter).

On the other hand, 4 to 5 Arabidopsis thaliana plant bodies were grownin 9 cm pots containing soil composed of equivalent portions ofvermiculite and perlite, for 6 weeks. Then, the Arabidopsis thalianaplant body was directly immersed in the Agrobacterium suspension of theAgrobacterium C58 (pBI35S: OsDREB1A) and placed in a desiccator, whichwas sucked with a vacuum pump to reduce the pressure to 650 mmHg andthen left to stand for 10 min. Subsequently, the plant pot wastransferred to a tray and covered with a wrap to maintain humidity. Onthe next day, the wrap was removed. Thereafter, the plant was grownuncovered to produce seeds. After sterilization in an aqueous solutionof sodium hypochlorite, the seeds were sown on an agar medium forselection (MS medium supplemented with 100 mg/l vancomycin and 30 mg/lkanamycin). Arabidopsis thaliana grown on this medium were transferredto pots to obtain seeds of the transformed plant.

3. Gene Introduction into Rice by Agrobacterium Infection

Rice seeds were immersed in 70% ethanol for 1 minute and sterilized byimmersion into 2% sodium hypochlorite for 1 hour. The sterilized seedswere then washed with sterilized water, and 9 grains each of the seedswere sowed onto a plate of N6D solid medium (3.98 g of CHU[N₆] BasalSalt Mixture (Sigma), 30 g of sucrose, 100 mg of myo-inositol, 300 mg ofcasamino acid, 2,878 mg of L-proline, 2 mg of glycine, 0.5 mg ofnicotinic acid, 0.5 mg of pyridoxine hydrochloride, 1 mg of thiaminehydrochloride, 2 mg of 2,4-D, and 4 g of Gelrite, per liter, pH 5.8),followed by culturing for 24 days. Thus, callus was induced. Thecalluses formed from approximately 20 grains of the seeds weretransferred to new N6D solid medium, followed by culturing foradditional three days.

Separately, Agrobacterium EHA105 (G-ubi: OsDREB1A) and AgrobacteriumEHA105 (G35S-ShD: OsDREB1A) were cultured in 5 ml of YEP mediumcontaining 100 mg/l rifampicilin and 20 mg/l kanamycin (10 g of Bactopeptone, 10 g of Bacto yeast extract, 5 g of NaCl, and 406 mg ofMgCl₂·6H₂O, per liter, pH 7.2) at 28° C. for 24 hours. ThisAgrobacterium was diluted with AAM medium containing 20 mg/lacetosyringon (10 mg of MnSO₄·5H₂O, 3 mg of H₃BO₃, 2 mg of ZnSO₄·7H₂O,250 μg of Na₂MoO₄·2H₂O, 25 μg of CuSO₄·5H₂O, 25 μg of CoCl₂·6H₂O, 750 μgof KI, 150 mg of CaCl₂·2H₂O, 250 mg of MgSO₄·7H₂O, 40 mg of Fe-EDTA, 150mg of NaH₂PO₄·2H₂O, 1 mg of nicotinic acid, 10 mg of thiaminehydrochloride, 1 mg of pyridoxine hydrochloride, 100 mg of myo-inositol,176.7 mg of L-arginine, 7.5 mg of glycine, 900 mg of L-glutamine, 300 mgof aspartic acid, and 3 g of KCl, per liter, pH 5.2) to bring O.D.₆₆₀ to0.1. Thus, 20 ml of Agrobacterium suspension was prepared.

Subsequently, to the callus, which was cultured for 3 days, theAgrobacterium suspension was added and then mixed for 1 minute.Thereafter, this callus was placed on a sterilized paper towel to removeexcess Agrobacterium suspension and then cultured on 2N6-AS solidmedium, on which the sterilized filter paper was placed, (3.98 g ofCHU[N₆] Basal Salt Mixture, 30 g of sucrose, 10 g of glucose, 100 mg ofmyo-inositol, 300 mg of casamino acid, 2 mg of glycine, 0.5 mg ofnicotinic acid, 0.5 mg of pyridoxine hydrochloride, 1 mg of thiaminehydrochloride, 2 mg of 2,4-D, 10 mg of acetosyringon, and 4 g ofGellite, per liter, pH 5.2) at 25° C. for 3 days in the dark. Afterculturing for 3 days, the culture product was thoroughly washed with anaqueous solution of 3% sucrose containing 500 mg/l carbenicillin untilthe product did not whiten. The washed culture product was furthercultured on N6D solid medium containing 500 mg/l carbenicillin and 10mg/l hygromycin for 1 week. Thereafter, the resulting culture productwas transferred onto a N6D solid medium containing 500 mg/lcarbenicillin and 50 mg/l hygromycin and cultured for 18 days.Furthermore, the callus was transferred to a regeneration medium (4.6 gof Murashige and Skoog Plant Salt Mixture (Nihon Pharmaceutical Co.,Ltd), 30 g of sucrose, 30 g of sorbitol, 2 g of casamino acid, 100 mg ofmyo-inositol, 2 mg of glycine, 0.5 mg of nicotinic acid, 0.5 mg ofpyridoxine hydrochloride, 0.1 mg of thiamine hydrochloride, 0.2 mg ofNAA, 2 mg of kinetin, 250 mg of carbenicillin, 50 mg of hygromycin, and8 g of agarose, per liter, pH 5.8). The product was transferred to a newmedium every week and regeneration. Those having buds grown toapproximately 1 cm were transferred to a hormone-free medium (4.6 g ofMurashige and Skoog Plant Salt Mixture (Nihon Pharmaceutical Co., Ltd),30 g of sucrose, 2 mg of glycine, 0.5 mg of nicotinic acid, 0.5 mg ofpyridoxine hydrochloride, 0.1 mg of thiamine hydrochloride, 50 mg ofhygromycin, and 2.5 g of Gellite, per liter, pH 5.8). Plant bodies,which have grown to approximately 8 cm on the hormone-free medium, weretransferred to a pot containing synthetic particulate potting soil(Bonsol No. 1, Sumitomo Chemical Co., Ltd.) to allow the transgenicplant to produce seeds.

Example 4 Analysis of the Transcription Activating Mechanism Using RiceProtoplast

As shown in FIG. 5, to construct an effector plasmid, OsDREB1A cDNA,OsDREB2A cDNA, DREB1A cDNA, and DREB2A cDNA were positioned downstreamof the CaMV35S promoter and the intron sequence of sucrose synthetase ofmaize and ligated to pBI221 plasmid (Clontech). Separately, a reporterplasmid was constructed in which a 75 bp DNA fragment containing DRE ofrd-29A promoter was repeatedly inserted twice upstream of the minimalpromoter −61 rd29A and a GUS reporter gene.

Subsequently, these two plasmids were introduced into the riceprotoplast and GUS activity was then determined based on changes influorescence intensity caused by decomposition of4-methylumbelliferyl-β-D-glucuronide. The fusion gene of CaMV35Spromoter-LUC was simultaneously introduced as a standard for theintroduction efficiency in each experiment. As a result, OsDREB1A andOsDREB2A genes were found to activate transcription through DRE.

Example 5 Analysis of Expression of OsDREB Gene in Transformant

1. Analysis of Expression of OsDREB Gene in Nontransformant

Expression properties of OsDREB1A, OsDREB1B, OsDREB1C, and OsDREB2Agenes in wild-type rice were analyzed by Northern hybridization. Ricewas cultured hydroponically at 25° C. under insolation conditions of 16hours during the day and 8 hours at night for 17 days. Abscisic acid,dehydration, low temperature, salt (NaCl), lesion, and water stresseswere separately applied to the plant body. Sampling was accomplished onstress-applied rice every 0, 10, 20, 40, 60 minutes, 2, 5, 10, and 24hours.

Each stress was applied to the rice as follows: abscisic acid stress wasapplied by immersing in a solution containing 100 μM ABA; dehydrationstress was applied by drying on a filter paper; low temperature stresswas applied by transferring to an incubator which was cooled at 4° C.;salt (NaCl) stress was applied by immersing in an aqueous solutioncontaining 250 mM NaCl; lesion stress was applied by slitting up 8 to 10cm-high leaves; and water stress was applied by immersing in pure water.Total RNA was separately prepared from a control plant which was givenno stress and the plant which was given stress. The RNAs were thensubjected to electrophoresis. Thus, the expression of each gene wasobserved by the Northern method. The result is shown in FIG. 6.

From analysis, the expression of the OsDREB1A gene and that of theOsDREB1B gene were respectively induced mainly by low temperaturestress. In contrast, the expression of OsDREB2A was induced mainly bydehydration and salt stresses. Gene expression was constantly observedin OsDREB1C.

2. Analysis of OsDREB Gene Expression in Transformed Arabidopsisthaliana

In the same manner as in Example 3, transformants having OsDREB1A,OsDREB1D, and OsDREB2A genes introduced into Arabidopsis thaliana wereprepared. The mRNA level of the transformant-introduced genes OsDREB1A,OsDREB1D, and OsDREB2A and that of the genes, the expression of whichwas considered to be altered by the introduced genes, were analyzed bythe Northern method. Specifically, partial fragments of rd29A gene,cor15a gene, kin1 gene, and erd10 gene were used as probes (rd29A: SEQID NO: 19, cor15a: SEQ ID NO: 20, kin1 SEQ ID NO: 21, erd10: SEQ ID NO:22), and the mRNA levels were analyzed. In addition to the transformant,transformed Arabidopsis thaliana having pBI121 plasmid (Clontech)containing no DREB-homologous gene introduced therein was used as acontrol to compare the gene expressions.

Approximately 1 g of plant bodies grown on GM agar medium for 3 weekswas exposed to dehydration stress and low temperature stress.Dehydration stress was applied by uprooting the plant from the agarmedium and drying it on a petri dish for 5 hours. Low temperature stresswas applied by incubating the plant at 4° C. for 5 hours. Total RNA wasprepared separately from control plants which are given no stress andplants which were given the dehydration and low temperature stresses.The resultant total RNAs were subjected to electrophoresis. Then, geneexpressions were assessed by the Northern method.

Generally, genes are introduced into the genome of a transformant in asimilar manner, however, due to differences in the locations on thegenome and thereby the expression of the introduced genes vary. Thisphenomenon is called “position effect.” In this experiment, by assayingtransformants with DNA fragments from the introduced gene as a probe bythe Northern method, those transformants in which the introduced genewas expressed more highly could be selected. Also, by using a DNAfragment of the gene, which could be involved in the stress tolerance,as a probe, OsDREB1A was introduced. Thus, the gene having a variedlevel of mRNA was identified. The result is shown in FIG. 7.

As a result, the gene having GCCGAC in the promoter was induced morestrongly than the gene having ACCGAG In the group of stress tolerantgenes of monocotyledonous plants, those having GCCGAC as the DREsequence exist in a larger amount than those having ACCGAG. Accordingly,it is suggested that, in these monocotyledonous plants, the OsDREB genesallowed the stress tolerant genes to express more efficiently than theDREB genes.

3. Analysis of OsDREB Gene Expression in Transformed Rice

In the same manner as in Example 3, transformants having OsDREB1A,OsDREB1B, and DREB1C genes of Arabidopsis thaliana introduced into ricewere prepared. The mRNA level of the transformant-introduced genesOsDREB1A, OsDREB1B, and DREB1C of Arabidopsis thaliana and that of thegene, the expression of which was considered to be altered by theintroduced genes, were analyzed by the Northern method. Specifically,partial fragments of OsDREB1A gene, OsDREB1B gene, DREB1C gene, lip9gene, Wsi724 gene, and salT gene were used as probes (OsDREB1A: SEQ IDNO: 23, OsDREB1B: SEQ ID NO: 24, DREB1C: SEQ ID NO: 25, lip9: SEQ ID NO:26, Wsi724: SEQ ID NO: 27, salT: SEQ ID NO: 28), and the expressionlevels of mRNA were analyzed. In the analysis, in addition to thetransformant, transformed rice having G-ubi that contained noDREB-homologous gene introduced therein was used as a control in orderto compare gene expressions.

Selection was carried out in a 0.1% Benlate solution containing 30 mg/mlhygromycin for 5 days. Thereafter, the plant was transferred to a potcontaining Bonsol No. 1 and was grown for 12 days. Approximately 2 g ofthe grown plant was subjected to salt (NaCl) and low temperaturestresses. Salt stress was applied by uprooting plant body from the soiland immersing in 250 mM NaCl in a test tube for 5 hours. Low temperaturestress was applied by incubating the plant body at 4° C. for 5 hours.Total RNAs were separately prepared from a control plant which was givenno stress and the plant which was given salt and low temperaturestresses, and then subjected to electrophoresis. Thus, the expression ofeach gene was observed by the Northern method in the same manner as in(2). The result is shown in FIG. 8.

As a result, in the transformed rice having OsDREB1A, OsDREB1B, andDREB1C genes introduced therein, the expression of the lip9 gene havingthe DRE sequence in the promoter region was induced while the expressionof the salT gene having no DRE sequence in the promoter region was notinduced. Also, the expression of the Wsi724 gene, the expression ofwhich in the promoter region was not identified but deduced to be atarget of OsDREB, based on the expression pattern when stress wasapplied (dehydration, salt, low temperature inducible, induction by lowtemperature is slower than that by dehydration and salt), was induced inthese transformants.

Example 6 Influences of OsDREB Genes on Arabidopsis thaliana StressTolerances

In the same manner as in Example 3, transformants having OsDREB1A andDREB1A genes introduced into Arabidopsis thaliana were prepared. As acontrol, Arabidopsis thaliana, which was transformed with pBI121containing no DREB-homologous gene, was prepared. Each toleranceexperiment was carried out under following conditions.

1. NaCl Tolerance

NaCl tolerance was inspected as follows. Arabidopsis thaliana, which wasgrown in GM medium for 3 weeks, was immersed in an aqueous solution of600 mM NaCl for 2 hours, followed by washing. Thereafter, the plant bodywas transferred into a pot containing Professional potting soil andcultured for 3 weeks, and its survival rate was assessed.

2. Dehydration Tolerance

Dehydration tolerance was investigated as follows. Arabidopsis thaliana,which was grown in GM medium for 3 weeks, was transferred in a potcontaining soil composed of equivalent portions of vermiculete andperlite, and cultured for 1 week, and water supply was then stopped.After culturing for 2 weeks, its survival ratio was assessed.

3. Freezing Tolerance

Freezing tolerance was investigated as follows. Arabidopsis thaliana,which was grown in GM medium for 3 weeks, was transferred into a potcontaining Professional potting soil and cultured for 1 week.Thereafter, the plant body was placed at −6° C. for 36 hours and thencultured at 22° C. for 5 days. Its survival ratio was then assessed.

In the experiment for inspecting salt stress tolerance, the survivalratio was 12% for the control and 55% or 65% for the OsDREB1A-introducedplant. As for the DREB1A-introduced plant, the survival ratio was 68%for 35S: DREB1A and 90% for 29A: DREB1A. This indicates that the OsDREBgenes also improve stress tolerance in dicotyledonous plants (FIG. 9).

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

Free Text of Sequence Listing

SEQ ID NO: 16; probe

SEQ ID NO: 17; primer

SEQ ID NO: 18; primer

SEQ ID NO: 19; probe for rd29a

SEQ ID NO: 20; probe for cor15a

SEQ ID NO: 21; probe for kin1

SEQ ID NO: 22; probe for erd10

SEQ ID NO: 23; probe for OsDREB1A

SEQ ID NO: 24; probe for OsDREB1B

SEQ ID NO: 25; probe for DREB1C

SEQ ID NO: 26; probe for lip9

SEQ ID NO: 27; probe for Wsi724

SEQ ID NO: 28; probe for salT

1. An isolated nucleic acid molecule which comprises the nucleotidesequence as shown in SEQ ID NO: 3 encoding the OsDREB1B protein.
 2. Anisolated nucleic acid molecule encoding a protein which comprises theamino acid sequence as shown in SEQ ID NO:
 4. 3. A recombinant vectorcomprising the nucleic acid molecule according to claim
 1. 4. Anisolated host cell transformed with the recombinant vector according toclaim
 3. 5. A transgenic plant transformed with the recombinant vectoraccording to claim
 3. 6. A method for producing the OsDREB1B proteinwhich regulates the transcription of genes located downstream of astress responsive element, wherein the transgenic plant according toclaim 5 is cultured in a medium and said protein is recovered from theresultant culture product.
 7. The transgenic plant according to claim 5,wherein the plant is a monocotyledonous plant.
 8. A method for producingthe OsDREB1B protein which regulates the transcription of genes locateddownstream of a stress responsive element, wherein the transgenic plantaccording to claim 7 is cultured in a medium and said protein isrecovered from the resultant culture product.
 9. A recombinant vectorcomprising the nucleic acid molecule according to claim 1, operablylinked downstream of a stress responsive promoter.
 10. An isolated hostcell transformed with the recombinant vector according to claim
 9. 11. Atransgenic plant transformed with the recombinant vector according toclaim 9, wherein the plant is a monocotyledonous plant.
 12. A method forimproving the stress tolerance of plants by introducing the nucleic acidmolecule according to claim 1 into the plants.
 13. A recombinant vectorcomprising the nucleic acid molecule according to claim
 2. 14. Anisolated host cell transformed with the recombinant vector according toclaim
 13. 15. A transgenic plant transformed with the recombinant vectoraccording to claim
 13. 16. The transgenic plant according to claim 15,wherein the plant is a monocotyledonous plant.
 17. A method forproducing the OsDREB1B protein which regulates the transcription ofgenes located downstream of a stress responsive element, wherein thetransgenic plant according to claim 15 is cultured in a medium and saidprotein is recovered from the resultant culture product.
 18. A methodfor producing the OsDREB1B protein which regulates the transcription ofgenes located downstream of a stress responsive element, wherein thetransgenic plant according to claim 16 is cultured in a medium and saidprotein is recovered from the resultant culture product.
 19. Arecombinant vector comprising the nucleic acid molecule according toclaim 2, operably linked downstream of a stress responsive promoter. 20.An isolated host cell transformed with the recombinant vector accordingto claim
 19. 21. A transgenic plant transformed with the recombinantvector according to claim 19, wherein the plant is a monocotyledonousplant.
 22. A method for improving the stress tolerance of plants byintroducing the nucleic acid molecule according to claim 2 into theplants.